Medium throughput protocol for genome-based quantification of intracellular mycobacterial loads and macrophage survival during in vitro infection

Abstract

Summary: Here, we present a streamlined protocol for assessing intracellular Mycobacterium tuberculosis (Mtb) loads in macrophages. This protocol describes the simultaneous assessment of macrophage viability using automated microscopy. Further, we detail the quantification of mycobacterial loads using a rapid, inexpensive, and accurate approach for mycobacterial DNA isolation from paraformaldehyde-fixed macrophages. Simultaneous assessment of the bacterial loads using internal standard and macrophage viability allows for precise quantification of the effects of perturbations on Mtb and host cells while accounting for technical artifacts.For complete details on the use and execution of this protocol, please refer to Chatterjee et al. (2021).

Keywords

• Cell-based Assays
• Immunology
• Microbiology
• Microscopy
• Molecular Biology